Adheson variants

ABSTRACT

Novel derivatives of cell surface proteins which are homologous to the immunoglobulin superfamily (adhesons) are provided. Amino acid sequence variations are introduced into adheson, the most noteworthy of which are those in which the transmembrane and, preferably, cytoplasmic domains are rendered functionally inactive, and in which adheson extracellular domains replace an immunoglobulin variable region. These variants are useful in therapy or diagnostics, in particular, CD4 variants are therapeutically useful in the treatment of HIV infections.

[0001] This is a Continuation-in-Part of U.S. Ser. No. 07/104,329, filedOct. 2, 1987.

BACKGROUND OF THE INVENTION

[0002] This application relates to compositions for antiviral orimmunomodulatory therapy. In particular, it relates to compositionsuseful in the treatment of Human Immunodeficiency Virus (HIV)infections.

[0003] The primary immunologic abnormality resulting from infection byHIV is the progressive depletion and functional impairment of Tlymphocytes expressing the CD4 cell surface glycoprotein (H. Lane etal., Ann. Rev. Immunol. 3:477 [1985]). CD4 is a non-polymorphicglycoprotein with homology to the immunoglobulin gene superfamily (P.Maddon et al., Cell 42:93 [1985]). Together with the CD8 surfaceantigen, CD4 defines two distinct subsets of mature peripheral T cells(E. Reinherz et al., Cell 19:821 [1980]), which are distinguished bytheir ability to interact with nominal antigen targets in the context ofclass I and class II major histocompatibility complex (MHC) antigens,respectively (S. Swain, Proc. Natl. Acad. Sci. 78:7101 [1981]; E.Engleman et al., J. Immunol. 127:2124 [1981]; H. Spitz et al., J.Immunol. 129:1563 [1982]; W. Biddison et al., J. Exp. Med. 156:1065[1982]; and D. Wilde et al., J. Immunol. 131:2178 [1983]). For the mostpart, CD4 T cells display the helper/inducer T cell phenotype (E.Reinherz, supra), although CD4 T cells characterized ascytotoxic/suppressor T cells have also been identified (Y. Thomas etal., J. Exp. Med. 154:459 [1981]; S. Meuer et al., Proc. Natl. Acad.Sci. USA 79:4395 [1982]; and A. Krensky et al., Proc. Natl. Acad. Sci.USA 79:2365 [1982]). The loss of CD4 helper/inducer T cell functionprobably underlies the profound defects in cellular and humoral immunityleading to the opportunistic infections and malignancies characteristicof the acquired immunodeficiency syndrome (AIDS) (H. Lane supra).

[0004] Studies of HIV-I infection of fractionated CD4 and CD8 T cellsfrom normal donors and AIDS patients have revealed that depletion of CD4T cells results from the ability of HIV-I to selectively infect,replicate in, and ultimately destroy this T lymphocyte subset (D.Klatzmann et al., Science 225:59 [1984]). The possibility that CD4itself is an essential component of the cellular receptor for HIV-I wasfirst indicated by the observation that monoclonal antibodies directedagainst CD4 block HIV-I infection and syncytia induction (A. Dalgleishet al., Nature [London] 312:767 [1984]; J. McDougal et al., J. Immunol.135:3151 [1985]). This hypothesis has been confirmed by thedemonstration that a molecular complex forms between CD4 and gp120, themajor envelope glycoprotein of HIV-I (J. McDougal et al., Science231:382 [1986]; and the finding that HIV-I tropism can be conferred uponordinarily non-permissive human cells following the stable expression ofa CD4 cDNA (P. Maddon et al., Cell 47:333 [1986]). Furthermore, theneurotropic properties of HIV-I, reflected by a high incidence ofcentral nervous system dysfunction in HIV-I infected individuals (W.Snider et al., Ann. Neurol. 14:403 [1983]), and the ability to detectHIV-I in the brain tissue and cerebrospinal fluid of AIDS patients (G.Shaw et al., Science 227:177 [1985]; L. Epstein, AIDS Res. 1:447 [1985];S. Koenig, Science 233:1089 [1986]; D. Ho et al., N. Engl. J. Med.313:1498 [1985]; J. Levy et al., Lancet II:586 [1985]), appears to haveits explanation in the expression of CD4 in cells of neuronal, glial andmonocyte/macrophage origin (P. Maddon, Cell 47:444 [1986]; I. Funke etal., J. Exp. Med. 165:1230 [1986]; B. Tourvieille et al., Science234:610 [1986]).

[0005] In addition to determining the susceptibility to HIV-I infection,the manifestation of cytopathic effects in the infected host cellappears to involve CD4. Antibody to CD4 was found to inhibit the fusionof uninfected CD4 T cells with HIV-I infected cells in vitro; moreover,the giant multinucleated cells produced by this event die shortly afterbeing formed resulting in the depletion of the population of CD4 cells(J. Lifson et al., Science 232:1123 [1986]). Formation of syncytia alsorequires gp120 expression, and can be elicited by coculturingCD4-positive cell lines with cell lines expressing the HIV-I env gene inthe absence of other viral structural or regulatory proteins (J.Sodroski et al., Nature 322:470 [1986]; J. Lifson et al., Nature 323:725[1986]). Thus, in mediating both the initial infection by HIV-I as wellas eventual cell death, the interaction between gp120 and CD4constitutes one of several critical entry points in the viral life cycleamenable to therapeutic intervention (H. Mitsuya et al., Nature 325:773[1987]).

[0006] The known sequence of the CD4 precursor predicts a hydrophobicsignal peptide, an extracellular region of approximately 370 aminoacids, a highly hydrophobic stretch with significant identity to themembrane-spanning domain of the class II MHC beta chain, and a highlycharged intracellular sequence of 40 residues (P. Madden, Cell 42:93[1985]). The extracellular domain of CD4 consists of four contiguousregions each having amino acid and structural similarity to the variableand joining (V-J) domains of immunoglobulin light chains as well asrelated regions in other members of the immunoglobulin gene superfamily(a subclass of which are defined herein by the coined term “adhesons”.These structurally similar regions of CD4 are termed the V₁, V₂, V₃ andV₄ domains (denominated 1-4 in FIG. 3).

[0007] A successful strategy in the development of drugs for thetreatment of many receptor mediated abnormalities has been theidentification of antagonists which block binding of the natural ligand.Since the CD4 adheson ordinarily binds to the recognition sites of theHIV envelope it would appear to be a candidate for therapeuticallysequestering these HIV sites, thereby blocking viral infectivity.However, full length CD4 and other adhesons are cell membrane proteinswhich are anchored in the lipid bilayer of cells. The presence ofmembrane components will be undesirable from the standpoint ofmanufacturing and purification. In addition, since adhesons are normallypresent only on cell surfaces, it would be desirable to produce adhesonsin a form which is more stable in the circulation. Additionally, eventruncated, soluble CD4 adheson (generally referred to as CD4T) may notbe optimally effective as a therapeutic since it possesses a relativelyshort biological half-life, binds to HIV no better than cell surfaceCD4, may not cross the placental or other biological barriers and sinceit merely sequesters the HIV recognition sites without in itself bearingan infected-cell killing or virus killing functionality.

[0008] Accordingly, it is an object of this invention to producesoluble, secreted adhesons. It is another object to produce CD4derivatives useful in the treatment of AIDS and related conditions, in amanner essentially unaffected by the extreme degree of genetic variationobserved among various HIV-I isolates and their respective envpolypeptides (J. Coffin, Cell 46:1 [1986]). Still another object is toprepare adhesons fused to other polypeptides in order to providemolecules with novel functionalities such as those described above fortherapeutic use, or diagnostic reagents for the in vitro assay ofadhesons or their ligands. In particular, it is an objective to preparemolecules for directing toxins or effector molecules (for example the Fcdomain of immunoglobulin) to cells bearing receptors for the adhesons,e.g. HIV gp120 in the case of CD4, and for use in facilitatingpurification of the adhesons. It is a further object to provide stable,highly purified adheson preparations.

SUMMARY

[0009] The objects of this invention are accomplished by providingnucleic acid encoding an amino acid sequence variant of an adheson, inparticular a variant in which the trans-membrane domain is modified sothat it is no longer capable of becoming lodged in the cell membrane. Inthe case of CD4 such variants are termed soluble CD4.

[0010] Variant adhesons are produced by a method comprising (a)transforming a host cell with nucleic acid encoding an amino acidsequence variant of an adheson, (b) culturing the host cell and (c)recovering the variant adheson from the host cell culture media or fromlysates of the host cell.

[0011] In specific embodiments, the objects of this invention areaccomplished by providing an adheson variant selected from the groupconsisting of (a) an adheson amino acid sequence variant having aninactivated transmembrane domain and (b) a polypeptide comprising anadheson extracellular domain fused to the sequence of a polypeptidewhich is different from the adheson, this latter, for example, selectedfrom a cytotoxin, an immunogen or a protein with a long plasma half lifesuch as an immunoglobulin constant domain.

[0012] In a preferred embodiment a polypeptide comprising a gp120binding domain of the CD4 adheson is fused at its C-terminus to animmunoglobulin constant domain, or is linked to a cytotoxic polypeptidesuch as ricin.

[0013] The CD4 adheson variants provided herein are purified andformulated in pharmacologically acceptable vehicles for administrationto patients in need of antiviral, neuromodulatory or immunomodulatorytherapy, in particular patients infected with HIV, and for use in themodulation of cell adhesion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIGS. 1a-1 c depict the amino acid and nucleotide sequence of asecreted form of the CD4 adheson. The signal processing site isdesignated with an arrow.

[0015]FIGS. 2a-2 c depict the amino acid and nucleotide sequence of afusion of the herpes gD leader and N-terminal 27 residues to theputative mature N-terminus of CD4T.

[0016]FIG. 3 depicts the structural elements of the native and solubleCD4 adheson, the native human IgG₁ (γ₁) heavy chain and two exemplaryheavy chain-CD4 chimeras.

[0017]FIGS. 4a-4 b are a map of the Tinkered human IgG₁ (γ₁) chainfragment employed in the preparation of CD4 fusions. Insert sites aredesignated γ1 and Fc.

[0018]FIG. 5 is a map of the human κ light chain fragment useful for CD4fusions at the arrow flanked by V_(κ)J_(κ) (light variable and joining)and C_(κ) (light constant).

DETAILED DESCRIPTION

[0019] Adhesons are cell surface polypeptides having an extracellulardomain which is homologous to a member of the immunoglobulin genesuperfamily, excluding, however, highly polymorphic members of thissuperfamily selected from the group of class I and class II majorhistocompatibility antigens, immunoglobulins and T-cell receptor α, β, γand δ chains. Examples of adhesons include CD1, CD2, CD4, CD8, CD28, theγ, δ and ε chains of CD3, OX-2, Thy-1, the intercellular or neural celladhesion molecules (I-CAM or N-CAM), lymphocyte function associatedantigen-3 (LFA-3), neurocytoplasmic protein (NCP-3), poly-Ig receptor,myelin-associated glycoprotein (MAG), high affinity IgE receptor, themajor glycoprotein of peripheral myelin (Po), platelet derived growthfactor receptor, colony stimulating factor-1 receptor, macrophage Fcreceptor, Fc gamma receptors and carcinoembryonic antigen. Homologous asdefined herein means having the sequence of a member of theimmunoglobulin gene superfamily or having a sequence therewithin whichhas substantially the same as (or a greater degree of) amino acidsequence homology to a known member of the superfamily as the specificexamples given above have to the sequence of an immunoglobulin variableor constant domain. Preferred adhesons are CD4, CD8 and high affinityIgE Fc receptor.

[0020] This invention is particularly concerned with amino acid sequencevariants of adhesons. Amino acid sequence variants of adhesons areprepared with various objectives in mind, including increasing theaffinity of the adheson for its binding partner, facilitating thestability, purification and preparation of the adheson, increasing itsplasma half life, improving therapeutic efficacy as described above inthe background, introducing additional functionalities and lessening theseverity or occurrence of side effects during therapeutic use of theadheson. Amino acid sequence variants of adhesons fall into one or acombination of the following classes: insertional, substitutional ordeletional variants.

[0021] Insertional amino acid sequence variants are those in which oneor more amino acid residues extraneous to the adheson are introducedinto a predetermined site in the adheson including the C or N termini.Such variants are referred to as fusions of the adheson and a differentpolypeptide. Such other polypeptides contain sequences other than thosewhich are normally found in the adheson at the inserted position.Several groups of fusions are contemplated herein. Immunologicallyactive adheson fusions comprise an adheson and a polypeptide containinga non-adheson epitope. The non-adheson epitope is any immunologicallycompetent polypeptide, i.e., any polypeptide which is capable ofeliciting an immune response in the animal to which the fusion is to beadministered or which is capable of being bound by an antibody raisedagainst the non-adheson polypeptide. Typical non-adheson epitopes willbe those which are borne by allergens, autoimmune epitopes, or otherpotent immunogens or antigens recognized by pre-existing antibodies inthe fusion recipient, including bacterial polypeptides such as trpLE,beta-galactosidase, viral polypeptides such as herpes gD protein, andthe like. Immunogenic fusions are produced by cross-linking in vitro orby recombinant cell culture transformed with DNA encoding an immunogenicpolypeptide. It is preferable that the immunogenic fusion be one inwhich the immunogenic sequence is joined to or inserted into the adhesonantigen or fragment thereof by a peptide bond(s). These productstherefore consist of a linear polypeptide chain containing adhesonepitopes and at least one epitope foreign to the adheson. It will beunderstood that it is within the scope of this invention to introducethe epitopes anywhere within the adheson molecule or fragment thereof.Such fusions are conveniently made in recombinant host cells or by theuse of bifunctional cross-linking agents. The use of a cross-linkingagent to fuse the adheson to the immunogenic polypeptide is not asdesirable as a linear fusion because the cross-linked products are notas easily synthesized in structurally homogeneous form.

[0022] These immunogenic insertions are particularly useful whenformulated into a pharmacologically acceptable carrier and administeredto a subject in order to raise antibodies against the adheson, whichantibodies in turn are useful in diagnostics or in purification ofadheson by immunoaffinity techniques known per se. Alternatively, in thepurification of adhesons, binding partners for the fused non-adhesonpolypeptide, e.g. antibodies, receptors or ligands, are used to adsorbthe fusion from impure admixtures, after which the fusion is eluted and,if desired, the adheson is recovered from the fusion, e.g. by enzymaticcleavage.

[0023] Other fusions, which may or may not also be immunologicallyactive, include fusions of the adheson sequence with a signal sequenceheterologous to the adheson, fusions of transmembrane-modified CD4adhesons, for example, to polypeptides having enhanced plasma half life(ordinarily>about 20 hours) such as immunoglobulin chains or fragmentsthereof, and fusions with cytotoxic functionalities. Signal sequencefusions are employed in order to more expeditiously direct the secretionof the adheson. The heterologous signal replaces the native adhesonsignal, and when the resulting fusion is recognized, i.e. processed andcleaved by the host cell, the adheson is secreted. Signals are selectedbased on the intended host cell, and may include bacterial yeast,mammalian and viral sequences. The herpes gD glycoprotein signal issuitable for use in mammalian expression systems.

[0024] Plasma proteins which have enhanced plasma half-life longer thanthat of transmembrane modified CD4 include serum albumin,immunoglobulins, apolipoproteins, and transferrin. Preferably, theadheson-plasma protein fusion is not significantly immunogenic in theanimal in which it is used and the plasma protein does not causeundesirable side effects in patients by virtue of its normal biologicalactivity.

[0025] In a specific embodiment the adheson immunoglobulin-like domainwhich may be homologous either to the constant or to the variable regiondomains is conjugated with an immunoglobulin constant region sequence.The resulting products are referred to herein as immunoadhesons.Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture. For example, see U.S. Pat.No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982); EP120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Köhler et al. ,P.N.A.S. USA 77:2197 (1980); Raso et al., Cancer Res. 41:2073 (1981);Morrison et al., Ann. Rev. Immunol. 2:239 (1984); Morrison, Science229:1202 (1985); Morrison et al. , P.N.A.S. USA 81:6851 (1984); EP255,694; EP 266,663; and WO 88/03559. Reassorted immunoglobulin chainsalso are known. See for example U.S. Pat. No. 4,444,878; WO 88/03565;and EP 68,763 and references cited therein.

[0026] Ordinarily, the domains of adhesons that are homologous toimmunoglobulins and extracellular in their native environment are fusedC-terminally to the N-terminus of the constant region of immunoglobulinsin place of the variable region(s) thereof, retaining at leastfunctionally active hinge, CH2 and CH3 domains of the constant region ofan immunoglobulin heavy chain. This ordinarily is accomplished byconstructing the appropriate DNA sequence and expressing it inrecombinant cell culture. Immunoglobulins and other polypeptides havingenhanced plasma half life are fused to the extracellular or ligandbinding domains of other adhesons in the same fashion.

[0027] The boundary domains for the CD4 V-like regions (V1-V4) are,respectively, about 100-109, about 175-184, about 289-298, and about360-369 (based on the precursor CD4 amino acid sequence in which theinitiating met is −25; FIG. 1a). CD4 sequences containing any of the CD4V domains are fused to the immunoglobulin sequence. It is preferablethat the V1V2 or V1V2V3V4 be fused at their C-termini to theimmunoglobulin constant region. The precise site at which the fusion ismade is not critical; the boundary domains noted herein are for guidanceonly and other sites neighboring or within the V regions may be selectedin order to optimize the secretion or binding characteristics of theCD4. The optimal site will be determined by routine experimentation. Ingeneral, it has been found that the fusions are expressedintracellularly, but a great deal of variation is encountered in thedegree of secretion of the fusions from recombinant hosts. For instance,the following table demonstrates the various immunoglobulin fusions thathave been obtained by the method of this invention. In all examples ofCD4 immunoadhesons, the CD4 signal was used to direct secretion from 293cells. Lower case m represents murine origin, while the lower case hdesignates human origin. V and C are abbreviations for immunoglobulinvariable and constant domains respectively. The numerical subscriptsindicate the number of parenthetical units found in the designatedmultimer. It will be understood that the chains of the multimers arebelieved to be disulfide bonded in the same fashion as nativeimmunoglobulins. The CD4 immunoadhesons typically contained either thefirst N-terminal 366 residues of CD4 (CD4₄) or the first 180 N-terminalresidues of CD4 (CD4₂) linked at their C-terminus to the κ (light) chainor IgG1 heavy chain constant region (γ1). TABLE I Transfected GeneSecreted Product mV_(κ)C_(κ) mV_(κ)C_(κ) and/or (mV_(κ)C_(κ))₂mV_(γ1)C_(γ1) ND mV_(κ)C_(κ) + mV_(γ1)C_(γ1)(mV_(κ)C_(κ))₂(mV_(γ1)C_(γ1))₂ + mV_(κ)C_(κ) and/or (mV_(κ)C_(κ))₂hCD4-mC_(κ) hCD4-mC_(κ) and/or (hCD4-mC_(κ))₂ hCD4-mC_(γ1) NDhCD4-mC_(κ) + hCD4-mC_(γ1) (hCD4-mC_(κ))₂(hCD4-mC_(γ1))₂ + hCD4-mC_(κ)and/or (hCD4-mC_(κ))₂ hCD4-hC_(κ) hCD4-hC_(κ) and/or (hCD4-hC_(κ))₂hCD4-hC_(γ1) (hCD4-hC_(γ1))₂ hCD4-hC_(κ) + hCD4-hC_(γ1)(hCD4-hC_(κ))₂(hCD4-hC_(γ1))₂ + hCD4-hC_(κ) and/or (hCD4-hC_(κ))₂mV_(κ)C_(κ) + hCD4-hC_(γ1) (mV_(κ)C_(κ))₂(hCD4-hC_(γ1))₂ + mV_(κ)C_(κ)and/or (mV_(κ)C_(κ))₂

[0028] It is interesting to observe from this table that the CD4-humanheavy chain immunoadheson was secreted as a dimer whereas the analogousmurine construction was not detected (this not excluding theintracellular accumulation of the protein, however). The ability of thehCD4-hCγ1 transformants to produce heavy chain dimer was unexpectedsince previous work had suggested that immunoglobulin heavy chains arenot secreted unless the hosts are cotransformed with nucleic acidencoding both heavy and light chain (Valle et al., Nature 241:338[1981]). According to this invention, CD4-IgG immunoadheson chimeras arereadily secreted wherein the CD4 epitope is present in heavy chaindimers, light chain monomers or dimers, and heavy and light chainheterotetramers wherein the CD4 epitope is present fused to one or morelight or heavy chains, including heterotetramers wherein up to andincluding all four variable region analogues are derived from CD4. Wherelight-heavy chain non-CD4 variable domain is present, a heterofunctionalantibody thus is provided.

[0029] Various exemplary hetero- and chimeric immunoadheson antibodiesproduced in accordance with this invention are schematically diagrammedbelow. “A” means at least a portion of the extracellular domain of anadheson containing its ligand binding site; V_(L), V_(H), C_(L) andC_(H) represent light or heavy chain variable or constant domains of animmunoglobulin; n is an integer; and Y designates a covalentcross-linking moiety.

[0030] (a) AC_(L);

[0031] (b) AC_(L)-AC_(L);

[0032] (c) AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)];

[0033] (d) AC_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)];

[0034] (e) AC_(L)-V_(H)C_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)];

[0035] (f) V_(L)C_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)]; or

[0036] (g) [A-Y]_(n)-[V_(L)C_(L)-V_(H)C_(H)]₂.

[0037] The structures shown in this table show only key features, e.g.they do not show joining (J) or other domains of the immunoglobulins,nor are disulfide bonds shown. These are omitted in the interests ofbrevity. However, where such domains are required for binding activitythey shall be construed as being present in the ordinary locations whichthey occupy in the adheson, immunoadheson or immunoglobulin molecules asthe case may be. These examples are representative of divalentantibodies; more complex structures would result by employingimmunoglobulin heavy chain sequences from other classes, e.g. IgM. Theimmunoglobulin V_(L)V_(H) antibody combining site also designated as thecompanion immunoglobulin, preferably is capable of binding to apredetermined antigen.

[0038] Suitable companion immunoglobulin combining sites and fusionpartners are obtained from IgG-1, -2, -3, or -4 subtypes, IgA, IgE, IgDor IgM, but preferably IgG-1.

[0039] A preferred embodiment is a fusion of an N-terminal portion ofCD4, which contains the binding site for the gp120 envelope protein ofHIV, to the C-terminal F_(c) portion of an antibody, containing theeffector functions of immunoglobulin G₁. There are two preferredembodiments of this sort; in one, the entire heavy chain constant regionis fused to a portion of CD4; in another, a sequence beginning in thehinge region just upstream of the papain cleavage site which defines IgGF_(c) chemically (residue 216, taking the first residue of heavy chainconstant region to be 114 [Kobat et al., “Sequences of Proteins ofImmunological Interest” 4th Ed., 1987], or analogous sites of otherimmunoglobulins) is fused to a portion of CD4. These embodiments aredescribed in the examples.

[0040] More particularly, those variants in which one or moreimmunoglobulin-like domains of an adheson are substituted for thevariable region of an immunoglobulin chain are believed to exhibitimproved in vivo plasma half life. These chimeras are constructed in afashion similar to chimeric antibodies in which a variable domain froman antibody of one species is substituted for the variable domain ofanother species. See, for example, EP 0 125 023; Munro, Nature 312:(Dec. 13, 1984); Neuberger et al., Nature 312: (Dec. 13, 1984); Sharonet al., Nature 309: (May 24, 1984); Morrison et al., Proc. Natl. Acad.Sci. USA 81:6851-6855 (1984); Morrison et al. Science 229:1202-1207(1985); and Boulianne et al., Nature 312:643-646 (Dec. 13, 1984). TheDNA encoding the adheson immunoglobulin-like domain(s) is cleaved by arestriction enzyme at or proximal to the 3′ end of the DNA encoding theimmunoglobulin-like domain(s) and at a point at or near the DNA encodingthe N-terminal end of the mature adheson polypeptide (where use of adifferent leader is contemplated) or at or proximal to the N-terminalcoding region for the adheson (where the native adheson signal isemployed). This DNA fragment then is readily inserted into DNA encodingan immunoglobulin light or heavy chain constant region and, ifnecessary, tailored by deletional mutagenesis. Preferably, this is ahuman immunoglobulin when the variant is intended for in vivo therapyfor humans. DNA encoding immunoglobulin light or heavy chain constantregions is known or readily available from cDNA libraries or issynthesized. See for example, Adams et al. , Biochemistry 19:2711-2719(1980); Gough et al., Biochemistry 19:2702-2710 (1980); Dolby et al.,P.N.A.S. USA, 77:6027-6031 (1980); Rice et al., P.N.A.S. USA79:7862-7865 (1982); Falkner et. al., Nature 298:286-288 (1982); andMorrison et al., Ann. Rev. Immunol. 2:239-256 (1984).

[0041] DNA encoding the immunoglobulin or immunoadheson chimericchain(s) is transfected into a host cell for expression. If the hostcell is producing an immunoglobulin prior to transfection then one needonly transfect with the adheson fused to light or to heavy chain toproduce a heteroantibody. The aforementioned immunoglobulins having oneor more arms bearing the adheson domain and one or more arms bearingcompanion variable regions result in dual specificity for adheson ligandand for an antigen. These are produced by the above-describedrecombinant methods or by in vitro procedures. In the latter case, forexample, F(ab′)₂ fragments of the adheson fusion and an immunoglobulinare prepared, the F(ab′)₂ fragments converted to Fab′ fragments byreduction under mild reducing conditions, and then reoxidized in eachother's presence under acidic conditions in accord with methods knownper se. See also U.S. Pat. No. 4,444,878.

[0042] Additionally, procedures are known for producing intactheteroantibodies from immunoglobulins having different specificities.These procedures are adopted for the in vitro production ofheterochimeric antibodies by simply substituting the immunoadhesonchains for one of the previously employed immunoglobulins.

[0043] In an alternative method for producing a heterofunctionalantibody, host cells producing an adheson-immunoglobulin fusion, e.g.transfected myelomas, also are fused with B cells or hybridomas whichsecrete antibody having the desired companion specificity for anantigen. Heterobifunctional antibody is recovered from the culturemedium of such hybridomas, and thus may be produced somewhat moreconveniently than by conventional in vitro resorting methods (EP68,763).

[0044] Another group of fusions are those in which an adheson isconjugated with a toxic substance, e.g. a polypeptide such as ricin(including deglycosylated ricin A chain), diptheria toxin A, or anon-peptidyl cytotoxin. Where the toxin is a polypeptide it isconvenient to cross-link the polypeptide to the adheson or itstransmembrane-deleted variant by conventional in vitro proteincross-linking agents (for suitable methods for linking ricin A chain ordeglycosylated A chain to CD4 see, for example, Duncan et al., “Analy.Biochem.” 132:68-73 [1983]; Thorpe et al., “Cancer Res.” 47:5924 [1987];and Ghotie et al., “Cancer Res.” 48:2610 [1988]) or by recombinantsynthesis as a fusion (see for example, U.S. Pat. No. 4,765,382).Alternatively, where companion antibodies are anti-ricin antibodyimmunoglobulin variable domains, such immunoglobulin heteroantibodiesare employed to deliver ricin to HIV infected cells following thegeneral procedure of Raso et al., Cancer Research, 41:2073 (1981).

[0045] Another class of adheson variants are deletional variants.Deletions are characterized by the removal of one or more amino acidresidues from a adheson sequence. Typically, the transmembrane andcytoplasmic domains of adhesons ar deleted. In the case of CD4, at leastresidues 368 to 395 (the transmembrane region), and ordinarily 396-433as well (the cytoplasmic domain), will be deleted to obtain secretedforms of this adheson. Parenthetically, the amino acid residues followthe numbers given for mature CD4 as noted, for example, in FIGS. 1a-1 c.Thus, CD4T molecules generally will terminate in the vicinity of aboutresidues 366-368, or at any other suitable site N-terminal thereto whichpreserves the gp120-binding capability of the CD4 variant.

[0046] Substitutional variants are those in which at least one residuein the adheson sequence has been removed and a different residueinserted in its place. The native N-terminal residue for mature CD4 isnow known to be lysine. Thus, the sequence shown in FIG. 1, with anN-terminal asparagine, is an amino acid sequence variant of nativemature CD4. Table 2 below describes substitutions which in general willresult in fine modulation of the characteristics of the CD antigen.TABLE 2 Original Residue Exemplary Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser; ala Gln asn Glu asp Gly pro His asn; gln Ileleu; val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyrSer thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

[0047] Substantial changes in function or immunological identity aremade by selecting substitutions that are less conservative than those inTable 2, i.e., selecting residues that differ more significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site or (c) the bulk of the side chain. The substitutionswhich in general are expected to produce the greatest changes in adhesonproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteinyl orprolyl is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanyl, is substituted for (or by) one not having a side chain,e.g., glycyl.

[0048] A preferred class of substitutional or deletional variants arethose involving the transmembrane region of the adheson. Thetransmembrane region of the adheson is a highly hydrophobic orlipophilic domain that is the proper size to span the lipid bilayer ofthe cellular membrane. It is believed to anchor the adheson in the cellmembrane.

[0049] Deletion or substitution of the transmembrane domain willfacilitate recovery and provide a soluble form of the adheson byreducing its cellular or membrane lipid affinity and improving its watersolubility. If the transmembrane and cytoplasmic domains are deleted oneavoids the introduction of potentially immunogenic epitopes, either byexposure of otherwise intracellular polypeptides that might berecognized by the body as foreign or by insertion of heterologouspolypeptides that are potentially immunogenic. A principal advantage ofthe transmembrane deleted adheson is that it is secreted into theculture medium of recombinant hosts. This variant is water soluble anddoes not have an appreciable affinity for cell membrane lipids, thusconsiderably simplifying its recovery from recombinant cell culture.

[0050] It will be amply apparent from the foregoing discussion thatsubstitutions, deletions, insertions or any combination thereof areintroduced to arrive at a final construct. As a general proposition, allvariants will not have a functional transmembrane domain and preferablywill not have a functional cytoplasmic sequence. This is generallyaccomplished by deletion of the relevant domain, although adequateinsertional or substitutional mutagens also can be effective for thispurpose. For example, the transmembrane domain is substituted by anyamino acid sequence, e.g. a random or homopolynucleic sequence of about5 to 50 serine, threonine, lysine, arginine, glutamine, aspartic acidand like hydrophilic residues, which altogether exhibit a hydrophilichydropathy profile, so that it is secreted into the culture medium ofrecombinant hosts. This variant should also be considered to be anadheson variant.

[0051] These variants ordinarily are prepared by site specificmutagenesis of nucleotides in the DNA encoding the adheson, therebyproducing DNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture. However, variant adhesons also are prepared byin vitro synthesis. Obviously, variations made in the DNA encoding thevariant adhesons must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure deleterious to expression (EP 75,444A). The CD4variants typically exhibit the same gp120 binding activity as does thenaturally-occurring prototype, although variants also are selected inorder to modify the characteristics of the CD4 adheson as indicatedabove.

[0052] While the site for introducing an amino acid sequence variationis predetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed adheson variants screened for the optimal combinationof desired activities. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis.

[0053] Adheson variants that are not capable of binding HIV gp120 areuseful nonetheless as immunogens for raising antibodies to the adhesonor as immunoassay kit components (labelled, as a competitive reagent forgp120 assay, or unlabelled as a standard for an adheson assay) so longas at least one adheson epitope remains active.

[0054] The DNA encoding adhesons is obtained by known procedures. SeeWilliams, Immunol. Today 8:298-303 (1987) and citations therein. Ingeneral, prokaryotes are used for cloning of CD4 variant DNA sequences.For example, E. coli strain SR101 (for propagating m13 phage, aλ-resistant strain of JM 101; Messing et al., Nucl. Acids. Res.9(2):309-321 [1981]); and E. coli K12 strain 294 (ATCC No. 31446) areparticularly useful. Other microbial strains which may be used includeE. coli B, UM101 and E. coli _(χ)1776 (ATCC No. 31537). These examplesare illustrative rather than limiting.

[0055] DNA encoding the variant adhesons are inserted for expressioninto vectors containing promoters and control sequences which arederived from species compatible with the intended host cell. The vectorordinarily, but need not, carry a replication site as well as one ormore marker sequences which are capable of providing phenotypicselection in transformed cells. For example, E. coli is typicallytransformed using a derivative of pBR322 which is a plasmid derived froman E. coli species (Bolivar, et al., Gene 2: 95 [1977]). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid must also contain or be modified to contain promotersand other control elements commonly used in recombinant DNAconstructions.

[0056] Promoters suitable for use with prokaryotic hosts illustrativelyinclude the β-lactamase and lactose promoter systems (Chang et al.,Nature, 275: 615 [1978]; and Goeddel et al., Nature 281: 544 [1979]),alkaline phosphatase, the tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res. 8: 4057 [1980] and EPO Appln. Publ. No. 36,776) andhybrid promoters such as the tac promoter (H. de Boer et al., Proc.Natl. Acad. Sci. USA 80: 21-25 [1983]). However, other functionalbacterial promoters are suitable. Their nucleotide sequences aregenerally known, thereby enabling a skilled worker operably to ligatethem to DNA encoding the adheson variant using linkers or adaptors tosupply any required restriction sites (Siebenlist et al., Cell 20: 269[1980]). Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantigen.

[0057] In addition to prokaryotes, eukaryotic microbes such as yeastcultures also are useful as cloning or expression hosts. Saccharomycescerevisiae, or common baker's yeast is the most commonly used eukaryoticmicroorganism, although a number of other strains are commonlyavailable. For expression in Saccharomyces, the plasmid YRp7, forexample, (Stinchcomb, et al., Nature 282: 39 [1979]; Kingsman et al,Gene 7: 141 [1979]; Tschemper et al., Gene 10: 157 [1980]) is commonlyused. This plasmid already contains the trp1 gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones,Genetics 85: 12 [1977]). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectivemeans of selection by growth in the absence of tryptophan.

[0058] Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.255: 2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7: 149 [1968]; and Holland, Biochemistry 17: 4900 [1978]),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

[0059] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., European Patent Publication No. 73,657A. Yeastenhancers also are advantageously used with yeast promoters.

[0060] Promoters for controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. the beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication. Fiers et al., Nature, 273: 113 (1978). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment. Greenaway, P. J. et al., Gene 18:355-360 (1982). Of course, promoters from the host cell or relatedspecies also are useful herein.

[0061] DNA transcription in higher eukaryotes is increased by insertingan enhancer sequence into the vector. Enhancers are cis-acting elementsof DNA, usually from about 10 to 300 bp, that act to increase thetranscription initiation capability of a promoter. Enhancers arerelatively orientation and position independent having been found 5′(Laimins, L. et al., Proc.Natl.Acad.Sci. 78: 993 [1981]) and 3′ (Lusky,M. L., et al., Mol. Cell Bio. 3: 1108 [1983]) to the transcription unit,within an intron (Banerji, J. L. et al., Cell 33: 729 [1983]) as well aswithin the coding sequence itself (Osborne, T. F., et al., Mol. CellBio. 4: 1293 [1984]). Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0062] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human or nucleated cells) may also containsequences necessary for the termination of transcription which mayaffect mRNA expression. These regions are transcribed as polyadenylatedsegments in the untranslated portion of the mRNA encoding the adheson.

[0063] Expression vector systems generally will contain a selectiongene, also termed a selectable marker. Examples of suitable selectablemarkers for mammalian cells are dihydrofolate reductase (DHFR),thymidine kinase or neomycin. When such selectable markers aresuccessfully transferred into a mammalian host cell, the transformedmammalian host cell can survive if placed under selective pressure.There are two widely used distinct categories of selective regimes. Thefirst category is based on a cell's metabolism and the use of a mutantcell line which lacks the ability to grow independent of a supplementedmedium. Two examples are: CHO DHFR⁻ cells and mouse LTK⁻ cells. Thesecells lack the ability to grow without the addition of such nutrients asthymidine or hypoxanthine. Because these cells lack certain genesnecessary for a complete nucleotide synthesis pathway, they cannotsurvive unless the missing nucleotides are provided in a supplementedmedium. An alternative to supplementing the medium is to introduce anintact DHFR or TK gene into cells lacking the respective genes, thusaltering their growth requirements. Individual cells which were nottransformed with the DHFR or TK gene will not be capable of survival innon supplemented media.

[0064] The second category is dominant selection which refers to aselection scheme used in any cell type and does not require the use of amutant cell line. These schemes typically use a drug to arrest growth ofa host cell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982), mycophenolic acid, Mulligan,R. C. and Berg, P. Science 209: 1422 (1980) or hygromycin, Sugden, B. etal., Mol. Cell. Biol. 5: 410-413 (1985). The three examples given aboveemploy bacterial genes under eukaryotic control to convey resistance tothe appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolicacid) or hygromycin, respectively.

[0065] “Amplification” refers to the increase or replication of anisolated region within a cell's chromosomal DNA. Amplification isachieved using a selection agent e.g. methotrexate (MTX) whichinactivates DHFR. Amplification or the making of successive copies ofthe DHFR gene results in greater amounts of DHFR being produced in theface of greater amounts of MTX. Amplification pressure is appliednotwithstanding the presence of endogenous DHFR, by adding ever greateramounts of MTX to the media. Amplification of a desired gene can beachieved by cotransfecting a mammalian host cell with a plasmid having aDNA encoding a desired protein and the DHFR or amplification genepermitting cointegration. One ensures that the cell requires more DHFR,which requirement is met by replication of the selection gene, byselecting only for cells that can grow in the presence of ever-greaterMTX concentration. So long as the gene encoding a desired heterologousprotein has cointegrated with the selection gene replication of thisgene gives rise to replication of the gene encoding the desired protein.The result is that increased copies of the gene, i.e. an amplified gene,encoding the desired heterologous protein express more of the desiredheterologous protein.

[0066] Preferred host cells for expressing the CD antigen variants ofthis invention are mammalian cell lines, examples including: monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293, Graham, F. L. et al., J. Gen Virol. 36: 59[1977] and 293S cells [293 subclones selected for better suspensiongrowth]); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamsterovary-cells-DHFR(CHO, Urlaub and Chasin, Proc.Natl.Acad.Sci. (USA) 77:4216, [1980]); mouse sertoli cells (TM4, Mather, J. P., Biol. Reprod.23: 243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); africangreen monkey kidney cells (VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51 cells); and TRI cells (Mather, J. P. etal., Annals N.Y. Acad. Sci. 383: 44-68 [1982]).

[0067] “Transformation” means introducing DNA into an organism so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integration. One suitable for transformation of the hostcells is the method of Graham, F. and van der Eb, A., Virology 52:456-457 (1973). However, other methods for introducing DNA into cellssuch as by nuclear injection or by protoplast fusion may also be used.If prokaryotic cells or cells which contain substantial cell walls areused as hosts, the preferred method of transfection is calcium treatmentusing calcium chloride as described by Cohen, F. N. et al., Proc. Natl.Acad. Sci. (USA), 69: 2110 (1972).

[0068] Construction of suitable vectors containing the desired codingand control sequences employ standard and manipulative ligationtechniques. Isolated plasmids or DNA fragments are cleaved, tailored,and religated in the form desired to form the plasmids required.Suitable procedures are well known for the construction describedherein. See, for example, (Maniatis, T. et al., Molecular Cloning,133-134 Cold Spring Harbor, [1982]; “Current Protocols in MolecularBiology”, edited by Ausubel et al., [1987], pub. by Greene PublishingAssociates & Wiley-Interscience).

[0069] Correct plasmid sequences are confirmed by transforming E. coliK12 strain 294 (ATCC 31446) with ligation mixtures, successfultransformants selected by ampicillin or tetracycline resistance whereappropriate, plasmids from the transformants prepared, and then analyzedby restriction enzyme digestion and/or sequenced by the method ofMessing et al., Nucleic Acids Res. 9: 309 (1981) or by the method ofMaxam et al., Methods in Enzymology 65: 499 (1980).

[0070] Host cells are transformed with the expression vectors of thisinvention. Thereafter they are cultured in appropriate culture media,e.g. containing substances for inducing promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

[0071] The secreted adheson variants are recovered and purified from theculture supernatants or lysates of recombinant hosts. Typically, thesupernatants are concentrated by ultrafiltration, contacted with aligand affinity or immunoaffinity matrix so as to adsorb the adhesonvariant, and eluted from the matrix. Optionally, the adheson is purifiedby ion exchange chromatography.

[0072] Surprisingly, purification of soluble CD4 adheson from culturemedium was unexpectedly difficult. Notwithstanding that the hydrophobictransmembrane region of the antigen had been deleted, the antigenexhibited a strong tendency to form aggregates that could be readilyremoved from suspension by centrifugation at 1000× g, and which avidlycoat surfaces such as ultrafiltration membranes. This appears to resultfrom the reduction in concentration of albumin or other serum protein(ordinarily present in the crude preparation) to a particular level,below which the truncated antigen no longer remains soluble. Thisphenomenon appears to be aggravated by exposure of the CD4 adheson tolow pH (<about pH 4). As a result, separation procedures (particularlythose that employ acid elution, such as immunoaffinity) should bemodified so that the eluate is maintained at, or immediately returnedto, about neutrality. Further, a surfactant, e.g. a detergent such asTween 80, should be included with the antigen during the separationprocedure. The final purified product will be stabilized with apredetermined protein such as albumin, and/or a detergent.

[0073] The purified adheson is formulated into conventionalpharmacologically acceptable excipients.

[0074] It is administered to patients having HIV infection at a dosagecapable of maintaining a concentration of greater than about 100 ng ofsoluble CD4 adheson/ml plasma. For CD4 adheson variants having differentmolecular weights, about 2 picomoles of soluble receptor per ml ofplasma will be initially evaluated clinically in order to establish astoichiometric equivalence with native (membrane bound) and solublereceptor. The ordinary dosage of soluble CD4 is 100 μg/kg of patientweight/day.

[0075] The therapeutic CD4 variants are employed with other therapiesand agents for the treatment of AIDS, including AZT, neutralizingantibodies and immunocytotoxins, gp120 fragments and vaccines.

[0076] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0077] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0078] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0079] “Recovery” or “isolation” of a given fragment of DNA from arestriction digest means separation of the digest on polyacrylamide oragarose gel by electrophoresis, identification of the fragment ofinterest by comparison of its mobility versus that of marker DNAfragments of known molecular weight, removal of the gel sectioncontaining the desired fragment, and separation of the gel from DNA.This procedure is known generally (Lawn, R. et al., Nucleic Acids Res.9: 6103-6114 [1981], and Goeddel, D. et al., Nucleic Acids Res. 8: 4057[1980]).

[0080] “Dephosphorylation” refers to the removal of the terminal 5′phosphates by treatment with bacterial alkaline phosphatase (BAP). Thisprocedure prevents the two restriction cleaved ends of a DNA fragmentfrom “circularizing” or forming a closed loop that would impedeinsertion of another DNA fragment at the restriction site. Proceduresand reagents for dephosphorylation and other recombinant manipulationsare conventional. Reactions using BAP are carried out in 50 mM Tris at68° C. to suppress the activity of any exonucleases which may be presentin the enzyme preparations. Reactions were run for 1 hour. Following thereaction the DNA fragment is gel purified.

[0081] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T. et al.,Id. at 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 μg of approximately equimolar amounts of the DNAfragments to be ligated.

[0082] “Filling” or “blunting” refers to the procedures by which thesingle stranded end in the cohesive terminus of a restrictionenzyme-cleaved nucleic acid is converted to a double strand. Thiseliminates the cohesive terminus and forms a blunt end. This process isa versatile tool for converting a restriction cut end that may becohesive with the ends created by only one or a few other restrictionenzymes into a terminus compatible with any blunt-cutting restrictionendonuclease or other filled cohesive terminus. Typically, blunting isaccomplished by incubating 2-15 μg of the target DNA in 10 mM MgCl₂, 1mM dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) buffer at about 37°C. in the presence of 8 units of the Klenow fragment of DNA polymerase 1and 250 μM of each of the four deoxynucleoside triphosphates. Theincubation generally is terminated after 30 min. phenol and chloroformextraction and ethanol precipitation.

[0083] The following examples merely illustrate the best mode nowcontemplated for practicing the invention, but should not be construedto limit the invention. All literature citations herein are expresslyincorporated by reference.

EXAMPLE 1 Construction of Vectors for the Expression of Native CD4 andSecreted Derivatives

[0084] Section 1

[0085] The plasmid used for recombinant synthesis of human CD4 waspSVeCD4DHFR. The plasmid was constructed as follows:

[0086] λCD4P1 containing most of the coding sequence of human CD4(obtained from a human placental cDNA library using oligonucleotideprobes based on the published sequence [Maddon et al. 1985]) wasdigested with EcoRI to produce the cDNA insert. This fragment wasrecovered by polyacrylamide gel electrophoresis (fragment 1).

[0087] pUC18 was digested with EcoRI and the single fragment recoveredby polyacrylamide gel electrophoresis (fragment 2). Fragment 1 wasligated to fragment 2 and the ligation mixture transformed into E. colistrain 294. The transformed culture was plated on ampicillin mediaplates and resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct DNA fragments. This plasmid is referred to as pUCCD4.

[0088] pSVeE′DHFR (Muesing et al., Cell 48:691-701 [1987]) was digestedwith KpnI and BamHI and blunted with E. coli DNA polymerase I (Klenowfragment) and the four dNTPs. Fragment 3 containing the pML-Amp^(r)region, SV40 early promoter, the HIV LTR, and the mouse DHFR gene wasrecovered by gel electrophoresis, ligated and the ligation mixturetransformed into E. coli strain 294. The transformed culture was platedon ampicillin media plates and resistant colonies selected. Plasmid DNAwas prepared from transformants and checked by restriction analysis forthe presence of the BamHI restriction site and the absence of the KpnIrestriction site. This plasmid is referred to as pSVeΔBKDHFR and allowsEcoRI-BamHI fragments to be inserted after the SV40 early promoter andtranscribed under its control, following transfection into anappropriate cell line.

[0089] Synthetic oligonucleotides (adaptors 1-8, below) were made toextend from 76 bp 5′ of the initiation codon of CD4 translation to theRsaI restriction site at 121 bp 3′ of the initiator, with the sequenceAATT at the 5′ end of the sense strand to generate an end which couldligate to an EcoRI restriction fragment. These oligonucleotides wereligated and the 204 bp fragment containing the entire sequence recoveredby gel electrophoresis (fragment 4). CD4 adaptor 1:AATTCAAGCCCAGAGCCCTGCCATTTCTGTGGGCTCAGGTCCCT CD4 adaptor 2:pACTGCTCAGCCCCTTCCTCCCTCGGCAAGGCCACAATGAACCGGGGAGT C CD4 adaptor 3:pCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGC CD4 adaptor 4:pAGCCACTCAGGGAAACAAAGTGGTGGTGGGCAAAAAAGGGGATACAGTG GAACTGACCTCT CD4adaptor 5: pACAGGTCAGTTCCACTGTATCCCCTTTTTTGCCCAGCACCACTTTGTTT CC CD4adaptor 6: pCTGAGTGGCTGCTGGGAGGAGCGCCAGTTGCAGCACCAGAAGCAAGT CD4 adaptor7: pGCCTAAAAGGGACTCCCCGGTTCATTGTGGCCTTGCCGAGGGAGGAAGG G CD4 adaptor 8:GCTGAGCAGTAGGGACCTGAGCCCACAGAAATGGCAGGGCTCTGGGCTTG

[0090] pUCCD4 was digested with RsaI and SstI and the 401 bp fragmentcontaining part of the CD4 coding sequence recovered by gelelectrophoresis (fragment 5). pUC18 was digested with EcoRI and SstI andthe fragment comprising the bulk of the plasmid recovered by gelelectrophoresis (fragment 6). Fragments 4 and 5 were ligated to fragment6 and the ligation mixture transformed into E. coli strain 294. Thetransformed culture was plated on ampicillin media plates and resistantcolonies selected. Plasmid DNA was prepared from transformants andchecked by restriction analysis for the presence of the correctfragment. The sequence of the inserted synthetic DNA was checked byexcising the 605 bp EcoRI-SstI fragments from several transformants andligating them to M13 mp19 which had been digested with the same enzymes.After transformation into E. coli strain JM101, single-stranded DNA wasprepared and sequenced. One plasmid which contained the correct sequencewas selected, and is referred to as pCD4int.

[0091] pCD4int was digested with EcoRI and SstI and fragment 7containing the 5′ end of the CD4 coding region was recovered by gelelectrophoresis. pUCCD4 was digested with SstI and BamHI and the 1139 bpfragment containing the remainder of the CD4 coding region (fragment 8)recovered by gel electrophoresis.

[0092] pSVeΔBKDHFR was digested with EcoRI and BamHI and fragment 9comprising the bulk of the plasmid was isolated. Fragments 7, 8 and 9were ligated and the ligation mixture transformed into E. coli strain294. The transformed culture was plated on ampicillin media plates andthe resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. This plasmid is referred to as pSVeCD4DHFR, andwas used to direct synthesis of recombinant intact CD4.

[0093] Section 2

[0094] A plasmid was constructed to direct the synthesis of a CD4derivative lacking the putative transmembrane domain and most of theputative cytoplasmic domain (Maddon et al.). This was done with theintention of creating a secreted form of CD4, based on the assumptionthat these domains anchor the CD4 glycoprotein to the cell membrane, andthat their deletion would result in the secretion of the product. Thisplasmid is referred to as pSVeCD4ΔNlaDHFR and was constructed asfollows:

[0095] pUCCD4 was digested with SstI and TaqI and the 531 bp fragment(fragment 10) recovered. pUCCD4 was digested with NlaIII and TaqI andthe 112 bp fragment (fragment 11) recovered. pUCCD4 was digested withBamHI and NlaIII and the 301 bp fragment (fragment 12) recovered.pCD4int was digested with SstI and BamHI and fragment 13 comprising thebulk of the plasmid recovered. Fragments 10, 11, and 12 were ligatedtogether with fragment 13 and the ligation mixture transformed into E.coli strain 294. The transformed culture was plated on ampicillin mediaplates and resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. Plasmid DNA from several transformants wassequenced to ensure that the 195 bp NlaIII fragment had been deleted andthat the proper reading frame was restored. The resulting plasmid isreferred to as pCD4ΔNla.

[0096] pCD4ΔNla was digested with EcoRI and BamHI and the 1541 bpfragment containing the sequence of a CD4 derivative lacking thetransmembrane and cytoplasmic domains recovered (fragment 14) andligated to fragment 9 and the ligation mixture transformed into E. colistrain 294. The transformed culture was plated on ampicillin mediaplates and resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. This plasmid is referred to as pSVeCD4ΔNlaDHFR.

[0097] Both pSVeCD4DHFR and pSVeCD4ΔNlaDHFR were transfected into CHOcells by the same method used to establish cell lines stably expressingHIV-I polypeptides (Muesing, Smith and Capon, Cell 48:6910701 [1987]).These cells were assayed for production by radioimmunoprecipitation asdescribed below. While no product was detected in initial experiments,subsequent experiments showed that the above described coding segmentcould indeed direct the synthesis of a soluble CD4 adheson variant bothin CHO and 293 cells.

[0098] Section 3

[0099] A different expression system was initially used for thesynthesis and expression of a CD4 variant lacking completely thecytoplasmic and transmembrane domains. This system uses thecytomegalovirus promoter and can be used in cultured cells of humanorigin. The first plasmid constructed for use in this system containedthe entire coding region for CD4 and was intended to function as acontrol in the following studies. It is referred to as pRKCD4, and wasconstructed as follows:

[0100] pSVeCD4DHFR was digested with EcoRI and BamHI and fragment 15containing the entire CD4 coding region was isolated. pRK5 (U.S. Ser.No. 97,472, filed Sep. 11, 1987) was digested with EcoRI and BamHI andfragment 16 comprising the bulk of the plasmid recovered by gelelectrophoresis, ligated to fragment 15, and the ligation mixturetransformed into E. coli strain 294. The transformed culture was platedon ampicillin media plates and resistant colonies selected. Plasmid DNAwas prepared from transformants and checked by restriction analysis forthe presence of the correct fragment. This plasmid is referred to aspRKCD4.

[0101] Section 4

[0102] The next plasmid constructed was designed to direct theexpression of the above-mentioned (Section 3) secreted derivative ofCD4. The coding region of CD4 was fused after amino acid residue 368 ofmature CD4 to a sequence from pBR322 which codes for 9 more residuesbefore a translation termination codon. This removes the putative CD4transmembrane and cytoplasmic domains, which are presumed to anchor CD4to the cell surface. The plasmid is referred to as pRKCD4T (and whichproduces protein called CD4T), and was constructed as follows:

[0103] pSVeCD4DHFR was digested with HpaIII, blunted with Klenowfragment and the four dNTPs, and digested with BstEII. The 382 bpfragment (fragment 17) containing part of the CD4 coding sequence wasrecovered by gel electrophoresis. pSVeCD4DHFR was digested with EcoRIand BstEII and the 874 bp fragment (fragment 18) recovered. pBR322 wasdigested with HindIII, blunted with Klenow fragment and the four dNTPs,and digested with EcoRI. Fragment 19 comprising the bulk of the plasmidwas isolated and ligated to fragments 17 and 18 and the ligation mixturetransformed into E. coli strain 294. The transformed culture was platedon ampicillin media plates and resistant colonies selected. Plasmid DNAwas prepared from transformants and checked by restriction analysis forthe presence of the correct fragment. This plasmid is referred to aspCD4Tint.

[0104] pRK5 was digested with EcoRI and SmaI and fragment 20 comprisingthe bulk of the plasmid isolated. pCD4Tint was digested with EcoRI andEcoRV and the 1410 bp fragment containing the CD4 coding sequence to theHpaII site at 1176 bp 3′ of the initiating codon and the 154 bpHindIII-EcoRV fragment of pBR322 was recovered (fragment 21). Fragments20 and 21 were ligated and the ligation mixture transformed into E. colistrain 294. The transformed culture was plated on ampicillin mediaplates and resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. This plasmid is referred to as pRKCD4T.

[0105] Section 5a

[0106] In order to create a secreted form of CD4 which could be purifiedwith an antibody directed to herpes virus type I glycoprotein D, aplasmid was constructed to express a derivative of CD4T in which theregion coding for the mature, processed CD4T polypeptide was fused to asequence coding for the signal peptide and the first 27 residues of themature type I Herpes Simplex Virus gD glycoprotein. This plasmid isreferred to as pRKGDCD4T, and was constructed as follows:

[0107] pgDTrunc.DHFR was digested with EcoRI and PvuII and the fragmentcontaining the coding region for the signal peptide and first 27residues of the mature HSV I gD glycoprotein was isolated (fragment 22).pRKCD4T was digested with EcoRI and BstEII and fragment 23 containingthe 3′ end of the CD4 coding sequence and the pRK5 region was isolated.

[0108] Synthetic oligonucleotides GD (adaptors 1-2, below) containingthe coding sequence of CD4 from the codon for the amino terminal residueof mature CD4 to the Rsa site at 121 bp 3′ of translation initiation,and containing the sequence CTGCTCGAG at the 5′ end of the sense strandwere prepared (fragment 24). pRKCD4 was digested with RsaI and BstEIIand the 665 bp fragment containing part of the coding region for CD4 wasrecovered (fragment 25) and ligated to fragment 24. After digestion withBstEII to ensure that only monomeric fragment was present, the 724 bpfragment containing both sequences was recovered by gel electrophoresis(fragment 26).

[0109] Fragments 22, 23 and 26 were ligated and the ligation mixturetransformed into E. coli strain 294. The transformed culture was platedon ampicillin media plates and resistant colonies selected. Plasmid DNAwas prepared from transformants and checked by restriction analysis forthe presence of the correct fragment. The sequence of severaltransformants was checked to ensure that the synthetic insert wascorrect and that reading frame was preserved. This plasmid is referredto as pRKGDCD4T.

[0110] These pRK5 derived plasmids preferably were transfected into 293Scells for stable expression according to Muesing, et al. Cell 48:691(1987) with the exception that in addition to the plasmid of interest aplasmid expressing the neomycin resistance gene pRSV neo (Gorman et al.Science 221:553-555 (1985)) was cotransfected. 293 cells also are usedsatisfactorily as host cells. 2 days after transfection, the cells werepassaged into standard medium (1:1 F12/DME supplemented withL-glutamine, penicillin-streptomycin and 10% FBS) with 0.5 mg/ml G418(Genticin sulfate; Gibco) for selection of stable cell lines, ratherthan in media containing methotrexate as shown by Muesing et al. Cellswere assayed for production of CD4 or CD4 analogs byradioimmunoprecipitation. Binding studies (section 5c) used conditionedsupernatants from these cells in the 1:1 F12/DME medium. Materials usedin infectivity assays (section 5b) were obtained as described in section8 below. gDCD4 adaptor 1:CTGCTCGAGCAGGGAAACAAAGTGGTGCTGGGCAAAAAGGGGATACAGTG GAACTGAC gDCD4adaptor 2: pACAGGTCAGTTCCACTGTATCCCCTTTTTTGCCCAGCACCACTTTGTTT CCCTGCTCGA

[0111] Section 5b

[0112] The following constitutes a study of the neutralization of HIV-1infectivity by soluble CD4 analogs. A modification of the neutralizationprocedure of Robert-Guroff et al., Nature 316:72 (1985) was followed.Equal volumes of inhibitor supernatant and virus (60 microliters) wereincubated at 4 degrees C. for 1 hour, then the same volume of H9 (Galloet al., Science 224:500, 1984) at 5×10⁶/ml was added and incubationcontinued for 1 hour at 37 degrees C. Following absorption, 2.5×10⁵cells in 150 microliters were transferred to 2 ml of incubation media.After 4 days at 37 degrees C., the cultures were split 1:2 with freshmedia and incubated for an additional 3 days. Cultures were harvested,reverse transcriptase activity was measured (Groopman et al., AIDSResearch and Human Retroviruses 3:71, 1987), and immunofluorescencereactivity with HIV-1 positive serum was determined as described (Poieszet al., Proc. Acad. Nat. Sci. USA 77:7415, 1980). Inhibitor supernatantswere obtained from confluent plate cultures of 293S/CDT4, 293S/gDCD4Tcells or untransfected 293S cells by replacing the growth mediumincubation media and harvesting the supernatants 24 hours later.Inhibitor supernatant replaced part or all of the incubation mediaduring the first three days of culture as indicated in the second columnof Table 3. Challenge dose of virus was 100 TCID₅₀ (Groopman et al.,supra) of HIV-1 strain HTLV-IIIB grown in H9 cells assayed in the samesystem. Incubation media consisted of RPMI 1640 media containing 2 mML-glutamine, 100 units/ml penicillin, 100 micrograms/ml streptomycin, 2micrograms/ml polybrene and 20% fetal calf serum (M.A. Bioproducts).TABLE 3 Dilution of Indirect Reverse Inhibitor Inhibitorimmunofluorescence transcriptase supernatant supernatant (% positivecells (cpm/ml × 10⁵) mock-trans- undil.; 1:4 65.3 65.5 21.8 23.9 fectedmock-trans- undil.; 1:4 61.2 61.1 18.5 28.1 fected CD4T undil.; 1:4 0.418.0 0.11 5.94 CD4T undil.; 1:4 0.8 16.1 0.15 3.72 gDCD4T undil.; 1:40.4 26.8 0.14 9.92 gDCD4T undil.; 1:4 1.4 36.1 0.23 11.3

[0113] Both forms of soluble CD4 virtually abolished the growth ofHIV-1, when incubated with virus-infected cells without prior dilution(Table 2). At a dilution of 1:4 the soluble CD4 preparations were onlypartially effective in inhibiting virus growth, however the level offluorescent-positive cells and reverse transcriptase was stillsignificantly lower than cultures receiving mock-transfected cellsupernatants (Table 2). Since there was no significant difference invirus growth between diluted and undiluted control supernatants, nor didany of the supernatants affect the growth of uninfected H9 cells (datanot shown), soluble CD4 proteins present in these supernatants wereconcluded to be responsible for the neutralization of HIV-1 infection ofH9 cells.

[0114] Section 5c

[0115] To determine the affinity constant for interactions between gp120and CD4 or CD4 variants, saturation binding analysis was carried outwith soluble CD4 (supra) and detergent solubilized intact CD4 (Lasky etal. Cell 50:975 [1987]) employing radioiodinated gp120 labeled withlactoperoxidase. Binding reactions consisted of ¹²⁵I-gp120 (3 ng to 670ng, 2.9 nCi/ng) incubated for 1 hour at 0 degrees C. with cell lysatescontaining intact CD4 (Laskey et al., op cit.) or cell supernatantscontaining unlabeled CD4T or gDCD4T prepared as described in section 5a.Reactions (0.2 ml) had a final composition of 0.5× McDougal Lysis Buffer(McDLB) (1× McDLB contains 0.5% Nonidet NP-40, 0.2% Na deoxycholate,0.12 M NaCl, 0.02 M Tris-HCl, pH 8.0) and were performed in duplicate,both in the presence or absence of 50 micrograms of unlabeled purifiedgp120 (74 fold or greater excess). Following incubation, bound gp120 wasquantitated by immunoprecipitation and counted in a gamma counter. Forimmunoprecipitation, binding reaction solutions were preabsorbed with 5microliters of normal rabbit serum for one hour at 0° C., and clearedwith 40 microliters of Pansorbin (10% w/v, Calbiochem) for 30 minutes at0 degrees C. Samples were then incubated overnight at 0 degrees C. with2 microliters of normal serum or 5 microliters (0.25 microgram) of OKT4monoclonal antibody (Ortho) followed by collection of immune complexeswith 10 microliters of Pansorbin. Precipitates were washed twice in 1×McDLB and once in water, then eluted by eluting at 100 degrees C. for 2minutes in sample buffer (0.12 M Tris-HCl pH 6.8, 4% SDS, 0.7 Mmercaptoethanol, 20% glycerol, and 0.1% bromophenol blue). CD4 moleculeswere bound saturably by gp120, and yielded a simple mass action bindingcurve. Supernatants from mock-transfected cells gave a level ofspecifically bound gp120 less than 1% that found for supernatantscontaining soluble CD4. Scatchard analysis revealed a single class ofbinding sites on each molecule, with apparent dissociation constants(Kd) of 1.3×10⁻⁹ M, 0.83×10⁻⁹ M and 0.72×10⁻⁹ M for intact CD4, CD4T andgDCD4T, respectively. The values obtained for CD4-gp120 binding insolution are comparable to the affinity previously measured for gp120binding to CD4 on whole cells (Kd-4.0×10⁻⁹ M. Lasky, Cell, supra).

[0116] Section 6

[0117] In order to produce secreted derivatives of CD4 which are free ofextraneous amino acid residues, two plasmids were constructed forexpression in 293 cells. The plasmids contain CD4 genes which have beentruncated without the addition of extra residues, and are referred to aspRKCD4ΔNla and pRKCD4TP (and which produce proteins called CD4TP andCD4ΔNla), and were constructed as follows:

[0118] Fragment 14 containing the CD4 gene with the 195 bp NlaIIIrestriction fragment deleted was ligated to fragment 16, which is pRK5digested with EcoRI and BamHI. The ligation mixture was transformed intoE. coli strain 294, the transformed culture plated on ampicillin mediaplates and resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. The resulting plasmid is referred to aspRKCD4ΔNla.

[0119] Synthetic DNA (5′ CGT GAT AGA AGC TTT CTA GAG 3′) was made toattach to the HpaII site at 1176 bp and which when so attached wouldterminate translation after amino acid residue 368 of mature CD4(fragment 27). The other end of this fragment was designed to ligate toBamHI restriction fragments. pUCCD4 was digested with BstEII and HpaIIand the 382 bp fragment containing part of the CD4 gene was recovered(fragment 28). Fragments 27 and 28 were ligated and then digested withBstEII to reduce dimerized fragments to monomers, and the resulting 401bp fragment was recovered (fragment 29).

[0120] pRKCD4 was digested with BstII and BamHI and the fragmentcomprising the bulk of the plasmid (fragment 30) was isolated andligated to fragment 29. The ligation mixture was transformed into E.coli strain 294, the transformed culture plated on ampicillin mediaplates and resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. The resulting plasmid is referred to as pRKCD4TP.Both plasmids are transfected into 293 cells to generate stable variantCD4-expressing cell lines as described above.

[0121] Section 7

[0122] Two plasmids were constructed to direct the expression ofsecreted CD4 lacking extraneous amino acid residues in CHO cells. Theseare referred to as pSVeCD4ΔNlaSVDHFR and pSVeCD4TPSVDHFR (and whichencode proteins having the primary sequence of CD4ΔNla and CD4TP), andwere constructed as follows:

[0123] pE348HBV.E400D22 was digested with PvuI and EcoRI and thefragment containing the SV40 early promoter and part of the β-lactamasegene was recovered (fragment 31). pE348HBV.E400D22 was digested withPvuI and BamHI and the large fragment containing the balance of theβ-lactamase gene as well as the SV40 early promoter and the DHFR genewas isolated (fragment 32).

[0124] Fragments 31 and 32 were ligated together with fragment 14 andtransformed into E. coli strain 294. The transformed culture was platedon ampicillin media plates and resistant colonies selected. Plasmid DNAwas prepared from transformants and checked by restriction analysis forthe presence of the correct fragment. The resulting plasmid is referredto as pSVECD4ΔNlaSVDHFR. This plasmid contains the same DNA fragmentencoding the soluble CD4 molecule found in the above-mentioned plasmidpSVeCD4ΔNlaDHFR (Section 2).

[0125] pRKCD4TP was digested with EcoRI and BamHI and the fragmentcontaining the truncated CD4 coding region was isolated and ligated tofragments 31 and 32. The ligation mixture was transformed into E. colistrain 294, the transformed culture plated on ampicillin media platesand resistant colonies selected. Plasmid DNA was prepared fromtransformants and checked by restriction analysis for the presence ofthe correct fragment. The resulting plasmid is referred to aspSVeCD4TPSVDHFR. Both of these plasmids are transfected into CHO cellsand amplified transfectants selected by methotrexate using conventionalprocedures.

EXAMPLE 2

[0126] Fusions of the V region of the CD4 gene, which is homologous tothe variable region of immunoglobulin genes (ref Maddon et al. 1985), tothe constant (C) region of human immunoglobulin κ and γ2 chains areconstructed as follows:

[0127] Synthetic DNA is made to code for the C region of human κ chain(residues 109-214) based on the sequence published by Morin et al.,Proc. Natl. Acad. Sci. 82:7025-7029, with the addition at the 5′ end ofthe coding strand of the sequence GGGG, which allows this fragment to beligated to the BspMI site at the end of the putative V-like region ofCD4. At the 3′ end of the coding region, a translational stop codon isadded as well as a sequence which allows this end to be ligated to BamHIrestriction fragments. The synthetic DNA is made in 8 fragments, 4 foreach strand, 70-90 bases long. These are then allowed to anneal andligated prior to isolation on a polyacrylamide gel (fragment 33).

[0128] pRKCD4 is digested with EcoRI and BspMI and the 478 bp fragmentcontaining the region coding for the putative V-like domain of CD4 isrecovered (fragment 34). Fragments 33 and 34 are ligated together withfragment 16 (from the expression vector pRK5). The ligation mixture istransformed into E. coli strain 294, the transformed culture plated onampicillin media plates and resistant colonies selected. Plasmid DNA isprepared from transformants and checked by restriction analysis for thepresence of the correct fragment. The resulting plasmid is referred toas pRKCD4Ck.

[0129] A plasmid encoding a fusion of the CD4 V-like domain to the humanimmunoglobulin Cγ2 region is constructed in a similar fashion, and isreferred to as pRKCD4Cγ2. Both of these plasmids are transfected into293 cells, myeloma cells or other competent cells in order to obtaincell lines expressing variant CD4 molecules as described above.

EXAMPLE 3

[0130] The gDCD4T secreted by the method of Example 1 was purified fromcell culture fluid containing either 10% FBS (fetal bovine serum) or noadded FBS. The conditioned cell culture fluid was first concentrated byultrafiltration then purified by immunoaffinity chromatography. Theimmunoaffinity column was produced by coupling murine monoclonalantibody 5B6 (whose epitope is on the HSV-1 gD portion of the gDCD4Tmolecule) to glyceryl coated controlled pore glass by the method of Royet al., 1984. The concentrated cell culture fluid is applied directly tothe column and the contaminating proteins are washed away with neutralpH buffer. The column is then washed with neutral buffer containingtetramethylammonium chloride followed by neutral buffer containing Tween80. The bound gDCD4T is eluted from the column with buffer at pH 3containing Tween 80 (0.1% w/v) and is neutralized immediately as it iseluted. The eluted neutralized gDCD4T is then concentrated byultrafiltration and dialyzed/diafiltered to exchange the buffer for aphysiological salt solution containing Tween 80 at approximately 0.1%w/v.

[0131] If the detergent is not present the gDCD4T forms aggregates asevidenced by the ability of centrifugation at approximately 10,000× gfor 2 minutes to remove the gDCD4T from the solution. Incubation ofgDCD4T at 4° C. in 0.1M sodium acetate, 0.5M NaCl and 0.25M tris at pH 7together with BSA, Tween 80 or glycerol as candidate stabilizers showedthat, in the absence of a stabilizer the gDCD4T gradually aggregatedover the space of 12 days to the point where only about 60-70% of theprotein was soluble. However, use of 0.1% w/v Tween 80 or (0.5 mg/ml BSAensured that about 100% or 80%, respectively, of the gDCD4T remainedsoluble over this period. Surprisingly glycerol was ineffective as astabilizer and produced results inferior even to the control—at 8 daysabout 80% of the gDCD4T was aggregated when stored in the presence ofglycerol.

EXAMPLE 4

[0132] Plasmids were constructed to direct the expression of proteinscontaining differing lengths of the amino-terminal, extracellular domainof CD4 fused to the constant region of human immunoglobulin γ1. Theseplasmids are referred to as pRKCD4_(2γ1), pRKCD4_(e4γ1), pRKCD4_(2γ1),pRKCD4_(e2γ1), pRKCD4_(1γ1), and pRKCD4_(e1γ1).

[0133] Plasmid pRKCD4_(4γ1) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for serinereside 366 of the mature CD4 polypeptide, immediately followed by thesequence coding for the constant region of human immunoglobulin γ1,starting at the codon for serine residue 114 of mature humanimmunoglobulin γ1 (Kabat et al.).

[0134] Plasmid pRKCD⁴ _(e4γ1) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for lysineresidue 360 of the mature CD4 polypeptide, immediately followed by thesequence coding for the constant region of human immunoglobulin γ1,starting at the codon for serine residue 114 of mature humanimmunoglobulin γ1 (Kabat et al.).

[0135] Plasmid pRKCD4_(2γ1) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for glutamineresidue 180 of the mature CD4 polypeptide, immediately followed by thesequence coding for the constant region of human immunoglobulin γ1,starting at the codon for serine residue 114 of mature humanimmunoglobulin γ1 (Kabat et al.).

[0136] Plasmid pRKCD4_(e2γ1) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for leucineresidue 177 of the mature CD4 polypeptide, immediately followed by thesequence coding for the constant region of human immunoglobulin γ1,starting at the codon for serine residue 114 of mature humanimmunoglobulin γ1 (Kabat et al.).

[0137] Plasmid pRKCD4_(1γ1) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for asparticacid residue 105 of the mature CD4 polypeptide, immediately followed bythe sequence coding for the constant region of human immunoglobulin γ1,starting at the codon for serine residue 114 of mature humanimmunoglobulin γ1 (Kabat et al.).

[0138] Plasmid pRKCD4_(e1γ1) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for leucineresidue 100 of the mature CD4 polypeptide, immediately followed by thesequence coding for the constant region of human immunoglobulin γ1,starting at the codon for serine residue 114 of mature humanimmunoglobulin γ1 (Kabat et al.).

[0139] Construction of these plasmids required the prior construction ofplasmid pRKCD4TP/γ1. It was constructed as follows:

[0140] A cDNA clone coding for human immunoglobulin γ1 was obtained froma human spleen cDNA library (Clontech Laboratories, Inc.) usingoligonucleotides based on the published sequence (Ellison et al., “Nucl.Acids Res.” 10:4071-4079 [1982]), and an EcoRI-EagI fragment (the EcoRIsite was contributed by a linker; see FIG. 4a, b) containing part of thevariable and all of the constant region was obtained. This fragment wasblunted with Klenow fragment, and recovered by gel electrophoresis(Fragment a1).

[0141] Plasmid pRKCD4TP-kk, encoding a substitutional variant of solubleCD4 (residues 1-368) containing a lysine residue instead of asparagineat position 1 of the mature polypeptide, was constructed from plasmidpRKCD4TP by site-directed mutagenesis. A synthetic oligonucleotide wasmade as a primer for a mutagenesis reaction to obtain the desired codingsequence. This was synthesized as a 51-mer which contained two silentmutations from the natural sequence in addition to the substitutionmutation, and 21 bases on each side of the mutated codons: 5′-CCC TTTTTT GCC CAG CAC CAC CTT CTT GCC CTG- AGT GGC TGC TGG GAG GAG-3′

[0142] Plasmid pRKCD4TP was transformed into E. coli strain SR101 andthe transformed colonies plated on ampicillin media plates. Resistantcolonies were selected and grown in the presence of m13K07 helperbacteriophage to yield secreted, encapsidated single-stranded templatesof pRKCD4TP. The single-stranded plasmid DNA was isolated and used asthe template for mutagenesis reactions with the syntheticoligonucleotides described above as primers. The mutagenesis reactionwas transformed E. coli SR101 and the transformed culture plated onampicillin media plates. Transformants were screened by colonyhybridization (ref. Grunstein-Hogness) for the presence of theappropriate sequence, using the following 16 mer as the probe.

[0143] 5′—C CAC CTT CTT GCC CTG—3′

[0144] The hybridization conditions chosen were sufficiently stringentthat the probe only detects the correctly fused product. Coloniesidentified as positive were selected and plasmid DNA was isolated andtransformed into E. coli strain SR101. The transformed cultures wereplated on ampicillin media plates, and resistant colonies were selectedand grown in the presence of m13K07 bacteriophage. Templates wereprepared as above and screened by sequencing.

[0145] Plasmid pRKCD4TP-kk was digested with XbaI and treated withKlenow Enzyme, and Fragment a2, containing the linearized plasmid wasrecovered by gel electrophoresis, and ligated with fragment al. Theligation mixture was transformed into E. coli strain 294, thetransformed culture plated on ampicillin media plates and resistantcolonies selected. Plasmid DNA was prepared from the transformants andchecked by restriction analysis for the presence of the correct fragmentin the correct orientation (i.e., the immunoglobulin coding region inthe same orientation as the CD4 coding region, and at the 3′ end of theCD4 coding region). This plasmid is referred to as pRKCD4TP/γ1.

[0146] Synthetic oligonucleotides were made as primers for deletionalmutagenesis reactions to fuse the appropriate coding sequences of IgG1and CD4 as described above. These were synthesized as 48-mers comprising24 nucleotides on each side of the desired fusion site (i.e.,corresponding to the COOH-terminal 8 residues of the desired CD4 moiety,and the NH₂-terminal 8 residues of the desired immunoglobulin moiety).Plasmid pRKCD4TP/γ1 was transformed into E. coli strain SR101 and thetransformed cultures plated on ampicillin media plates. Resistantcolonies were selected and grown in the presence of m13K07 helperbacteriophage to yield secreted, encapsidated single-stranded templatesof pRKCD4TP/γ1. The single-stranded plasmid DNA was isolated and used asthe template for mutagenesis reactions with the syntheticoligonucleotides described above as primers. The mutagenesis reactionswere transformed E. coli SR101 and the transformed culture plated onampicillin media plates. Transformants were screened by colonyhybridization (ref. Grunstein-Hogness) for the presence of theappropriate fusion site, using 16mers as probes. These 16mers comprise 8bases on either side of the fusion site, and the hybridizationconditions chosen were sufficiently stringent that the probes onlydetect the correctly fused product. Colonies identified as positive wereselected and plasmid DNA was isolated and transformed into E. colistrain SR101. The transformed cultures were plated on ampicillin mediaplates, and resistant colonies were selected and grown in the presenceof m13K07 bacteriophage. Templates were prepared as above and screenedby sequencing.

[0147] The plasmids were transfected into 293 cells using standardprocedures and assayed for expression and production as described above.Expressed Secreted pRKCD4_(1γ1) + − pRKCD4_(e2γ1) + + pRKCD4_(2γ1) + +pRKCD4_(e4γ1) + + pRKCD4_(4γ1) + +

[0148] Plasmids also were constructed to direct the expression of fusionproteins containing differing lengths of the amino-terminal,extracellular domain of CD4 fused to the truncated portion of theconstant region of human immunoglobulin γ1, comprising only the hingeregion and constant domains CH₂ and CH₃.

[0149] Synthetic oligonucleotides were made as primers for mutagenesisreactions to delete the immunoglobulin sequence from Ser114 to Cys215inclusive (Kabat et al.). These were synthesized as 48-mers comprising24 nucleotides on each side of the desired fusion site (i.e.,corresponding to the COOH-terminal 8 residues of the desired CD4 moiety,and the NH₂-terminal 8 residues of the desired immunoglobulin moiety).Plasmids pRKCD4_(4γ1), pRKCD4_(2γ1) and pRKCD4_(1γ1) were separatelytransformed into E. coli strain SR101 and the transformed culture platedon ampicillin media plates. Resistant colonies were selected and grownin the presence of m13K07 helper bacteriophage to yield secreted,encapsidated single-stranded templates of these plasmids. Thesingle-stranded plasmid DNA was isolated and used as the template formutagenesis reactions with the synthetic oligonucleotides describedabove as primers. The mutagenesis reactions were transformed E. coliSR101 and the transformed culture plated on ampicillin media plates.Transformants were screened by colony hybridization (Grunstein-Hogness)for the presence of the appropriate fusion site, using 16mers as probes.These 16mers comprise 8 bases on either side of the fusion site, and thehybridization conditions chosen were sufficiently stringent that theprobes only detect the correctly fused product. Colonies identified aspositive were selected and plasmid DNA was isolated and transformed intoE. coli strain SR101. The transformed cultures were plated on ampicillinmedia plates, and resistant colonies were selected and grown in thepresence of m13K07 bacteriophage. Templates were prepared as above andscreened by sequencing.

[0150] The plasmid derived from plasmid pRKCD4_(4γ1) is referred to aspRKCD4_(4Fc1), that derived from plasmid pRKCD4_(2γ1) is referred to aspRKCD4_(2Fc1) and that derived from plasmid pRKCD4_(1γ1) is referred toas pRKCD4_(1Fc1).

[0151] pRKCD4_(2Fc1), pRKCD4_(1Fc1) and pRKCD4_(4Fc1) are cultured inthe same fashion as described above and CH1-deleted CD4 immunoadhesonsrecovered as described elsewhere herein.

[0152] Light Chain Fusions

[0153] Plasmids were constructed to direct the expression of proteinscontaining differing lengths of the amino terminal, extracellular domainof CD4 fused to the constant region of human immunoglobulin κ. Theseplasmids are referred to as pRKCD4_(4κ), and pRKCD4_(e4κ).

[0154] Plasmid pRKCD4_(4κ) contains the portion of the CD4 gene from theinitiation codon to the fusion site after the codon for serine residue366 of the mature CD4 polypeptide, immediately followed by the sequencefor the constant region of human immunoglobulin κ, starting at the codonfor threonine residue 109 of the mature human immunoglobulin κ. (Kabatet al.)

[0155] Plasmid pRKCD4_(e4κ) contains the portion of the CD4 gene fromthe initiation codon to the fusion site after the codon for lysineresidue 360 of the mature CD4 polypeptide, immediately followed by thesequence for the constant region of human immunoglobulin κ, starting atthe codon for threonine residue 109 of the mature human immunoglobulinκ. (Kabat et al.)

[0156] These plasmids were constructed in a manner analogous to plasmidspRKCD4_(4γ1) and pRKCD4_(e4γ1) described above, with the followingexception:

[0157] The human immunoglobulin κ coding sequence (FIG. 5) was obtainedfrom a human spleen cDNA library (Clontech Laboratories, Inc.) usingoligonucleotides based on the published sequence (Hieter, P. A. et al.,Cell 22:197-207 [1980]) and an EcoRI-BspMI fragment containing part ofthe variable region and the entire constant region was obtained (seeFIG. 5). This fragment was blunted with Klenow fragment and the fourdNTPs. This fragment was used instead of fragment a1, and was used toconstruct plasmid pRKCD4TP/hκ.

[0158] Expression in CHO Cells

[0159] Plasmids were or are constructed to direct the expression of theimmunoadhesons described above in CHO cells. These are referred to aspSVeCD4_(4γ1)SVDHFR, pSVeCD4_(2γ1)SVDHFR, pSVeCD4_(1γ1)SVDHFR,pSVeCD4_(e4γ1)SVDHFR, pSVeCD4_(e2γ1)SVDHFR, pSVeCD4_(e1γ1)SVDHFR,pSVeCD4_(4Fc1)SVDHFR, pSVeCD4_(2Fc1)SVDHFR, pSVeCD4_(1Fc1)SVDHFR,pSVeCD4_(4κ)SVDHFR and pSVeCD4_(2κ)SVDHFR.

[0160] Fragment 31 was prepared as described above. Fragment 32a wasprepared by digesting plasmid pE348HBV.E400 D22 with BamHI, bluntingwith Klenow fragment and the four dNTPs, then digesting with PvuI andisolating the large fragment containing the balance of the β-lactamasegene and the SV40 early promoter and the DHFR gene. PlasmidspRKCD4_(4γ1), pRKCD4_(2γ1), pRKCD4_(1γ1), pRKCD4_(e4γ1), pRKCD4_(e2γ1),pRKCD4_(e1γ1), pRKCD4_(4Fc1), pRKCD4_(2Fc1), pRKCD4_(1Fc1), pRKCD4_(4κ)and pRKCD4_(2κ) were separately digested with HindIII, blunted withKlenow fragment and the four dNTPs, then digested with EcoRI and thefragments encoding the CD4-Ig fusion protein were isolated. Theresulting DNA fragments were ligated together with fragments 31 and 32aand transformed into E. coli strain 294. Colonies were selected andchecked for the presence of the correct plasmid as above, thentransfected into CHO cells and amplified by methotrexate selection usingconventional procedures.

EXAMPLE 5 Culture, Purification and Formulation of CD4 Variants

[0161] Plasmids encoding soluble CD4 adhesons such as CD4T, CD4TP, orsoluble CD4 immunoadhesons were calcium phosphate transfected intoCHO-DP7 (a proinsulin-transformed autocrine host cell derived from CHO;U.S. Ser. No. 97,472) and the transformants grown in selective medium(1:1 HAM F12/DMEM GHT⁻ containing 1-10% diafiltered or dialyzed bovineserum). Other suitable host cells are CHO cells or 293S human embryonickidney cells. The transformants were amplified by methotrexate selectionin the same medium but containing 500 nm methotrexate. A subclonecapable of secreting CD4TP, CD4tp 500 b, was selected. CD4tp 500 b iscultured in a DMEM/HAM F12 medium at about 37° C. until CD4TPaccumulates in the culture, after which the medium is separated from thecells and insoluble matter by centrifuging.

[0162] Culture fluid from CD4TP transformants was concentrated anddiafiltered to lower the ionic strength. The concentrate was passedthrough a large volume of Q-Sepharose anion exchange resin (previouslyequilibrated with 25 mM NaCl, pH 8.5) in order to adsorb contaminantsfrom the culture fluid. The isoelectric point of CD4TP is about 9.5,thus making it possible to discriminate between truncated forms of CD4and most contaminants by alternate adsorption, respectively, on a cationexchange resin such as carboxymethyl or sulfonyl Sepharose, and an anionexchange resin such as quaternary ammonium Sepharose. In addition, sincehighly electropositive domains are present in the extracellular segmentof CD4 any CD4-containing variant is purified in the same fashion asCD4TP. The unadsorbed culture fluid from the anion exchange resin stepwas then passed through a cation exchange resin (previously equilibratedwith 25 mM NaCl at pH 8.5) whereby CD4TP was adsorbed to the resin. TheCD4TP was eluted with a NaCl gradient at pH 8.5, this CD4 varianteluting at about 0.2 M NaCl. Ammonium sulfate was added to the eluate toa concentration of 1.7M and the solution passed through a column ofhydrophobic interaction chromatography resin (phenyl or butylSepharose). The CD4TP was eluted from the hydrophobic interaction columnwith a gradient of ammonium sulfate, the CD4TP emerging at about 0.7Mammonium sulfate. The eluate was concentrated and buffer exchanged on aG-25 column using phosphate buffered saline containing 0.02% (w/v) Tween20 or Tween 80. The CD4TP was soluble and stable in this solution, whichwas sterile filtered and filled into vials as an aqueous formulation.Other polymeric nonionic surfactants are suitably used with the CD4formulations, including Pluronic block copolymers or polyethyleneglycol.

[0163] It is also possible to employ immunoaffinity purification ofsoluble CD4 wherein the CD4 is adsorbed onto an immobilized antibodyagainst CD4. This method suffers from the disadvantage that elution ofthe soluble CD4 under acidic conditions leads to protein aggregationthat is only thoroughly ameliorated at relatively higher levels ofsurfactant. The foregoing procedure permits the use of much lowerquantities of surfactant, about from 0.01 to 0.10% (w/v) surfactant.

[0164] The procedure followed for the purification of CD4 fusions withimmunoglobulin heavy chain was to concentrate recombinant supernatantsby ultrafiltration and thereafter adsorb the fusion ontoresin-immobilized Staphylococcal protein A. The fusion was eluted with0.1M citrate buffer pH 3 with no salt or detergent. This preparation isbuffered into Tris buffer at pH 7.5. The immunoglobulin fusions with CD4V1-V4 optionally are further purified by the procedure described abovefor unfused CD4 variants. CD4 immunoglobulin fusions with CD4 V1-V2 alsomay be purified by the procedure above, except that it is not expectedthat the isoelectric point of this class of molecules will be asalkaline as that of species containing all four V regions of CD4.

EXAMPLE 6

[0165] The characteristics of several adheson variants were determined.As shown in table 4 the immunoadhesons CD4_(4γ1) and CD4_(2γ1) showimproved plasma half-life in rabbits, coupled with high-affinity gp120binding and an affinity for Fcγ receptor (determined with U937 cells)that is comparable to that of bulk human IgG1. TABLE 4 PlasmaHalf-Life⁺⁺ gp120 KD (nM)^(#) FcγR KD (nM)⁺ In Rabbits (Hrs.) CD4T^(§)2.3 ± 0.4 Not detected 0.25 CD4_(4γ1) 1.2 ± 0.1 2.83 ± 0.25 6.4CD4_(2γ1) 1.4 ± 0.1 3.01 ± 0.68 40.6 human IgG1 ND** 3.52 ± 0.5  21days*

1. Nucleic acid encoding an amino acid sequence variant of an adheson.2. The nucleic acid of claim 1 wherein the adheson is a CD4 polypeptide.3. The nucleic acid of claim 2 wherein the variant is a CD4 polypeptidein which nucleic acid encoding the transmembrane domain has beenmodified whereby the CD4 polypeptide encoded thereby contains aninactivated transmembrane domain.
 4. The nucleic acid of claim 3 whereinthe transmembrane domain has been inactivated by its deletion or bysubstituting for the transmembrane domain an amino acid sequence havinga substantially hydrophilic hydropathy profile.
 5. The nucleic acid ofclaim 2 wherein the variant comprises a fusion of (a) a polypeptidedifferent from the CD4 and (b) a CD4 polypeptide.
 6. The nucleic acid ofclaim 5 wherein the polypeptide different from the CD4 bears a non-CD4immune epitope.
 7. The nucleic acid of claim 6 wherein the polypeptidedifferent from CD4 is fused to the amino or carboxyl terminus of matureCD4 and the transmembrane domain of CD4 has been inactivated.
 8. Thenucleic acid of claim 5 wherein the different polypeptide comprises asignal sequence.
 9. The nucleic acid of claim 5 wherein the differentpolypeptide contains about from 5 to 1000 residues.
 10. The nucleic acidof claim 9 wherein the different polypeptide is capable of eliciting ahumoral immune response in an animal.
 11. The nucleic acid of claim 10wherein the different polypeptide is a viral polypeptide or an allergen.12. The nucleic acid of claim 5 wherein the different polypeptide is ahuman plasma protein having a plasma half life greater than from whichthe transmembrane domain has been deleted.
 13. The nucleic acid of claim12 wherein the variant is a fusion of a polypeptide comprising at leastone V-like domain of CD4 fused with a polypeptide comprising animmunoglobulin constant domain.
 14. The nucleic acid of claim 1 whereinthe adheson is CD4, CD8 or the high affinity IgE receptor.
 15. Thenucleic acid of claim 2 wherein the variant consists essentially of theV₁ through V₄ or V₁ through V₂ regions of the CD4 antigen.
 16. Thenucleic acid of claim 2 which consists essentially of the CD4 insert ofpCD4ΔNla.
 17. The nucleic acid of claim 12 wherein the differentpolypeptide is albumin, apolipoprotein or transferrin.
 18. The nucleicacid of claim 8 wherein the signal sequence is a bacterial signalsequence.
 19. The nucleic acid of claim 15 wherein the variant consistsessentially of CD4 residues 1-368.
 20. The nucleic acid of claim 15wherein the variant consists essentially of CD4 residues 1-180.
 21. Thenucleic acid of claim 13 wherein the immunoglobulin constant domain isthe constant domain of an IgG heavy chain.
 22. The nucleic acid of claim5 wherein the different polypeptide is a cytotoxic polypeptide.
 23. Thenucleic acid of claim 5 wherein the cytotoxic polypeptide is thediptheria toxin A.
 24. A composition comprising an adheson amino acidsequence variant which is incapable of cell membrane anchorage.
 25. Thecomposition of claim 24 wherein the adheson variant comprises a CD4amino acid sequence capable of binding gp120.
 26. The composition ofclaim 25 further comprising an agent for inhibiting the aggregation ofthe variant selected from the group of a predetermined protein and asurfactant.
 27. The composition of claim 26 wherein the agent is asurfactant.
 28. The composition of claim 27 wherein the surfactant isTween 80 or Tween
 20. 29. The composition of claim 25 wherein the CD4transmembrane domain has been deleted or has been substituted for by anamino acid sequence having a substantially hydrophilic hydropathyprofile.
 30. The composition of claim 29 which is sterile and whichfurther comprises a physiologically acceptable carrier.
 31. Thecomposition of claim 25 wherein the variant comprises an immunoglobulinamino acid sequence.
 32. The composition of claim 31 wherein theimmunoglobulin sequence comprises a constant domain sequence of animmunoglobulin heavy chain.
 33. The composition of claim 32 wherein theconstant domain is linked at its N-terminus to the C-terminus of atransmembrane-deleted CD4 polypeptide.
 34. The composition of claim 33wherein the CD4 polypeptide contains V₁V₂.
 35. The composition of claim33 wherein the CD4 polypeptide contains V₁V₂V₃V₄.
 36. The composition ofclaim 31 wherein the the variant is in the form of a dimer.
 37. Thecomposition of claim 36 wherein the composition comprises a fusion of aCD4 V-like domain to an immunoglobulin heavy chain constant domain. 38.The composition of claim 31 wherein the variant is selected from thegroup consisting of (a) AC_(L); (b) AC_(L)-AC_(L); (c) AC_(H)-[AC_(H),AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), orV_(L)C_(L)-V_(H)C_(H)]; (d) AC_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H),AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)]; (e)AC_(L)-V_(H)C_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)]; (f)V_(L)C_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)]; or (g)[A-Y]_(n)-[V_(L)C_(L)-V_(H)C_(H)]₂ wherein A is a CD4 polypeptidecontaining a CD4 variable region-like domain; V_(L), V_(H), C_(L) andC_(H) represent light or heavy chain variable or constant domains of animmunoglobulin; n is an integer; and Y designates the residue of acovalent cross-linking agent.
 39. The composition of claim 38 whereinthe V_(L) and V_(H) domains are capable of binding a predeterminedantigen.
 40. The composition of claim 31 wherein the immunoglobulinsequence is obtained from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD or IgM.41. The composition of claim 25 wherein the variant comprises apolypeptide different from CD4 which is nonimmunogenic in humans. 42.The composition of claim 41 wherein the variant comprises a polypeptidewhich is immunogenic in humans.
 43. The composition of claim 41 whereinthe variant comprises a polypeptide having a human plasma half lifewhich is greater than about 20 hours.
 44. The composition of claim 41wherein the variant comprises a human transferrin, apolipoprotein oralbumin polypeptide.
 45. The composition of claim 25 wherein the variantcomprises a cytotoxic polypeptide.
 46. The composition of claim 45wherein the cytotoxic polypeptide is ricin A chain or diptheria toxin A.47. A polypeptide comprising a CD4 amino acid sequence capable ofbinding gp120 which is cross-linked to (a) polypeptide having a plasmahalf life of greater than about 20 hours or (b) a cytotoxic polypeptide.48. The polypeptide of claim 47 wherein the polypeptide of (a) istransferrin, an apolipoprotein or albumin.
 49. The polypeptide of claim47 wherein the cytotoxic polypeptide is cross-linked to the CD4variable-like domain by a bifunctional cross-linking agent.
 50. A methodfor preparing an adheson variant comprising transfecting a host cellwith the nucleic acid of claim
 1. 51. A method for preparing an adhesonvariant comprising recovering the variant from the culture of a hostcell transfected with the nucleic acid of claim
 1. 52. The method ofclaim 51 wherein the adheson is CD4 and the variant is recovered fromthe culture medium of the host cell or from the cell itself.
 53. Themethod of claim 52 wherein the variant is recovered by adsorption onto acation exchange resin.
 54. The method of claim 53 wherein the variant isrecovered by adsorption of contaminants onto an anion exchange resin.55. The method of claim 52 wherein the variant lacks a functionaltransmembrane domain.
 56. The method of claim 52 wherein wherein a saltis added to the culture medium to occupy charged domains of the variant,the resulting solution is contacted with a hydrophobic affinitychromatography resin to adsorb the variant, and the variant eluted fromthe resin by washing the resin with a declining gradient of salt. 57.The method of claim 52 wherein the variant is recovered byimmunoaffinity chromatography.
 58. The method of claim 57 wherein theimmunoaffinity chromatography is directed against a polypeptidedifferent from CD4 which is fused to CD4.
 59. A method for the treatmentof an HIV infection comprising administering to a patient infected withHIV a therapeutically effective dose of an amino acid sequence variantof CD4.
 60. A replicable vector comprising the nucleic acid of claim 1.